11-oxygenated-14alpha-hydroxy-9alpha-halogen-pregnenes



This invention provides A -ll-oxygenated 14a-hydroxy9cz-ialogen-pregnenes and functional derivatives thereof.

The compounds unsubstituted in Zl-pcsition are more powerful thanprogesterone in transforming the vaginal epithelium into mucus cells inthe presence of estrone and therefore can be used therapeutically inthose illnesses where progesterone is indicated.

The compounds With oxygen in 2l-position have cortical hormone activity;they may be used in the alleviation of symptoms associated withrheumatic and arthritic discases.

The invention also provides a process for making the above new A-14-hydroxy-pregnenes, wherein an ll-oxygenated I l-unsubstituted A-9ot-halogen-pregnene is subjected to the aerobic action of an enzymeproduced by fungi of the genera Mucor, Helicostylum, Pleospora orCurvularia.

The ll-oxygenated l4-unsubstituted A -9u-halogen,especially-9ea-fiuoroand 9ot-chloro-pregnenes advantageously contain inthe 3- and -pc-sitions free or functionally converted hydroxyl or oxogroups. They may contain further double bonds, for example, in the 1-,6-, 7- or 16- position, or may contain additional substituents, such asfree or converted iydroxyl or oxo groups, and also epoxy groups orhalogen atoms, for example, in the 2-, 4-, 6-, 7-, 8-, 15-, 16-, l7- or2l-position, or methyl groups, for example, in the Hot-position. Theaforesaid starting materials may be of any desired steric configurationand may be used in the form of racemates. They also include those of theso-called norand/ or homo-series, especially l9-norand D-homo-compounds.Especially important starting materials are, for example, 9a-fluoro-and9achloro-dcrivatives of hydrocortisone, cortisone, corticosterone,lldehydrocorticosterone, corresponding l-dehydroand Zl-oxo-cornpounds,such as the 9efiuoroand 9achloro-derivativcs ofl-dehydro-hydrocortisone, l-dehydro-cortisone, l-dehydrocorticosterone,lzll-bisdehydrocorticosterone, ll-hydr xyand l1-oxo-progesterone, 11,8-hydroxyand ll-oxo-l7e-hydroxy-progesterone, l-dehyro-lLB-hydroxyandll-oxo-progesterone, l-dehydroll l7ct-dihydroxyand-1l-oxo-17ct-hydroxy-progesterone. In the starting materials thefunctionally converted hydroxyl group may be, for example, a hydroxylgroup esterified with aliphatic, aromatic or heterocyclic carboxylicacid, for example, acetic acid, propionic acid, benzoic acid or furanecarboxylic acid, or an etherified hydrox l group, for example, thetetrahydropyranyl-oxy-, benzyloxyor triphenylmethoxy-group. Thefunctionally converted oxo group is advantageously a ketalized oxo groupderived, more especially, from a dihydric alcohol such as theethylene-dioxy group.

The aforesaid starting materials are reacted in the process of theinvention with enzymes produced by fungi of the genus Mucor,Helicostylum, Pleospora or Curvularia, especially the species M HCOIgriseocyamts, Mucor parasitism, Helicostylum piriforme, Pleospomgaeumaimi and Cm'vularia pait'cscens. For cultivating these organismsknown methods may be used, for example, those using sugars such asglucose or lactose, peptones, corn steep liquor, soya bean products orthe like, and also mineral salts, or synthetic nutrient solutions. it isespecially advantageous to Work under aerobic conditions, for example,in an agitated culture, or with submerged growth While stirring and withthe access of air. The aforesaid moulds diller from othermicro-organisms, for example, bacteria, by growing Well under relativelysimple conditions of culture. The process of the invention can becarried out in the mould cultures described above or With the enzymecontained therein after being separated or, if desired, concentrated,and in the simplest case in a suspension of the separated mouldmyceliurn or of the homogenized mould mycelium or in a filtrate oraqueous extract therefrom.

The products of the process can be isolated, for example, by extractingthe reaction mixture with an organic solvent, for example, methylenechloride or ethyl acetate. For further purification of the extract soobtained chromatography is especially suitable, for example, overaluminum oxide or silica gel, or distribution methods may be used, forexample, the counter-current method, or separation may be carried out bymeans of Girard reagents, such as trimethylamrnoniumor pyridinium-aceticacid hydrazide. Instead of or in addition to the purification,recrystallization from an organic or aqueous-organic solvent may becarried out.

By introducing the l4-hydroxyl group there are obtained valuablell-oxygenated A -l4-hydroxy-9a-halogeru pregnenes and derivativesthereof, which, as compared with the therapeutically active compoundsnot hydroxylated in the l l position, are distinguished by their greateractivity. By oxygenated compounds and functional derivatives thereofthere are to be understood those which contain free or functionallyconverted hydroxyl or oxo groups such, for example, as esters, ethers,thioesters, thioethers, thioor thion esters, acetals, mercaptals,itetals, enol derivatives such as enol-esters, enol-ethers or enamines,and hydrazones, semicarbazones and toe lil' Among the products of theprocess thee may be n. tioned, more espec ally, the 9stiuoroand9u-c2hloro-deriyatives of l4ct-ny roxy-hydrocortisone,l4-crhydiox'='cortisone, l4ahydroxy-corticosterone, ll-whydroxy-ll-dehydro-corticosterone, 14c hydroxy 1dehydro-hydrocortisone, l4e-hydroxy-l-oenydro-cortisone, r -nydroxy-1-dehydro-corti osterone, ide-hydroxy-l ll-bisdehydro-corticosterone,lazllti-dih droxyand 1 .-1 droxy-ll-oxo hydroxy ll oxo ldehydroprogesterone, l hz:ll,6:l7a-..roxoand llmlh-dihydrox'-ll-oxo-ldehydroprogesterone, and also correspondingfunctional derivatives thereof, such esters or others. When the ro uctsof the process do ot possess the configura- -c and substituents or" sters useful for therapeutic pur roses, they can be used as intermediateproducts for the manufacture, example, of the above mentioned compounds.

"l he reaction products obtained by the process of the invention may beconverted by methods in themselves known into their functionalderivatives, such as oxygen, sulfur or nitrogen derivatives, forexample, esters, others, enol-esters, enol-cthers, ltetals, thioethersor thiolretals, and also hydrazones, oxirnes or enarnines, and hydroxylgroups may be dehydrogenated to form oxo groups. In these compounds thehydroxyl and/or xo groups may be completely or partially functionallyconverted.

in the esters and enol-esters the acid radicals may be base of anydesired organic or inor anic acids, such as aliphatic, all-cyclic,araliphatic, aromatic or heterocyclic carhoxylic acids, thion-carboxylicacids, thiol-carboxylic acids or sul'ionic 'ds, adv ageously formicacid, acetic acid, chloracetic acids, trifiuora acrea e acetic acid,caproic acids, oenanthic acids, caprylic acids, palmitic acids, crotonieacid, undecanic acid, undecylenic acid, oxalic acid, succinic acid,pimelic acid, maleic acid, lactic acid, carbamic acids,aikoxy-carboxylic acids, ,6- cyclopentyl-prop-ionic acid,hexahydrobenzoic acid, benzoic acid, phenylacetic acid, cyclohexylaceticacid, phenyl propionic acids, trimethyl-gallic acid, phthalic acid,furanc-Z-carboxylic acid, isonicotinic acid, methane sulfonic acid,toluene suli'onic acid, sulfuric acids, hydrohalic acids or phosphoricacids.

If desired, functionally converted hydroxyl or x0 groups present in thecompounds so obtained may be converted into free groups. In this manner,especially in polysubstituted derivatives the functionally convertedgroups may be only partially liberated. This may be brought about, forexample, by chemical or enzymatic hydrolysis, for example, with the useof acid or basic agents, by re-esterification or reacetalization. Fromthe only partially converted, such as esteriiied or etherified,derivatives obtained in this manner or obtained directly polysubstitutedderivatives, especially mixed esters or ethers or ester-others, can beproduced by subsequent functional conversion, for example,esterification or etherification. If, during the hydrolysis, especiallywhen carried out with an alkaline agent, the 9:11-halogen hydrin isconverted into the corresponding 9:11-oxidocompound, the latter can bereconverted into the desired 9:11-halogen hydrin by reaction with ahydrohalic acid, especially hydrofluoric acid or hydrochloric acid.

The products or" the invention are useful as medicaments or asintermediate products for making medicaments.

The following examples illustrate the invention:

Example 1 A solution of 125 milligrams of A -3:20dioxo-9a-fluoro-l 1(3:17a:ZI-trihydrOXy-pregnene in 10 cc. of acetone is added under sterileconditions to an agitation culture, which has been Well developed at 28C. and is four days old, of Plcospora gaeumanni in 500 cc. of aqueousbeer wort oi 70 percent strength containing 0.5 cc. of sperm oil. Thesuspension is agitated for 4 days at the same temperature. The myceliumis then separated and washed well with water and ethyl acetate. Thecombined clear solutions are extracttd with ethyl acetate. The ethylacetate solutions are washed with water, dried and evaporated in vacuo.The residue is dissolved in methanol of 80 percent strength, and thenextracted several times with petroleum ether. The methanol solutions arethen completely evaporated in vacuo. A paper chromatogram (mixture ofpropylene glycol and toluene) of the residue exhibits, in addition to asmall amount of A 3220-di0XO-9oc-fiuGlO-11B:174x121 trihydroxy pregnene,the somewhat slower running A -3:20-dioxo-9a-i'luoro- 116:14a:17a:21-tertrahydroxy-pregnene. The entire residue is split up bymeans of a preparative paper chromatogram (mixture of propylene glycoland toluene). The zones corresponding to the 14a-hydroxy-derivative arecut out and extracted several times with methanol of 50 percentstrength. The methanol is then removed in vacuo, the residual aqueoussolution is extracted several times with ethyl acetate, the combinedethyl acetate solutions are Washed with water, dried and evaporated, andpure A -3z20-dioxo-9ot-fluoro 11,8: 14a: 17u:21 tetrahydroxy-pregneneremains behind as a residue.

The incubation of the A -3:20-diox0-9ot-fluoro-ll5:l7ot:ZI-trihydroxy-pregnene may be carried out in 500 cc. of a welldeveloped aqueous culture of Cw'vnl'aria pallescens, which contains thefollowing additions: grams of cane sugar, 5 grams of Difco-tryptone, 1gram of sodium nitrate, 0.5 gram of secondary potassium orthophosphate,0.25 gram of sodium sulfate, 0.25 gram of potassium chloride, 5milligrams of iron sulfate hep-tallydrate, 1.25 grams of potassiumcarbonate and 0.5 cc. of sperm oil. The further treatment is asdescribed above.

Li -3:20-dioxo-9u-iluoro-116:14am 17a:21-tetrahydroxypregnene can beacetylated as follows: milligrams of this compound are mixed with 0.3cc. of acetic anhydride and 3 drops of pyridine. The solution is allowedto stand for 15 hours at 20 C., then evaporated on the addition of Waterunder ordinary vacuum and then completely dried under a high vacuum, andM6 :20-dioxo-9afiuoro-l 1 {3: 140a: 170a 21-tetrahy-droxy-pregncue-21acetate is obtained as a residue.

Example 2 A solution of milligrams of A -3:20-dioxo-9afiuoro-l 1 8:17az2l-trihydroxy-pregnadiene in 10 cc. of acetone is added understerile conditions to an agitation culture, which is well developed at28 C. and is 4 days old, of Pleospora gaeumamzi in 500 cc. of aqueousbeer wort of 70 per cent strength containing 0.5 cc. of sperm oil. Thesuspension is agitated for a further 4 days at the same temperature. Thereaction mixture is worked up and the resulting A"=-3:20-dioxo-9a-iluoro-1lfi:14a: l7az2l-tetrahydroxy-pregnadiene isisolated and purified as described in Example 1. V

The 11,82140421'7oc121 tetrahydroxy derivative so obtained travels moreslowly in a paper chromatogram (mixture of propylene glycol and toluene)than A -3 :20- dioxo-9a-fluoro-1L6: 17cc 21-trihydroxy-pregnadiene.

A -3 :20 dioxo 90c fluoro 11,6: a: :221-l6l1'2tl1Y- .droxy-pregnadienecan be acetylated in the manner described in Example 1 to yield A-3:2O-dioxo-9a-fluorol1 5: 14a:17a:21-tetrahydroxy-pregnadiene-Zl-acetate.

Example 3 wherein Y is selected from the group consisting of keto and,B-hydroxy, and R is selected from the group consisting of hydrogen andlower alkanoyl.

2. A compound selected from the group consisting of steroids of theformula (EHQR) (3:0

wherein Hal is a member selected from the group consisting of fluorineand chlorine, R is a member selected from the group consisting ofB-hydroxy and oxo groups and R is a member selected from the groupconsisting of hydrogen, hydroxy and acyloxy said acyloxy having 1 to 10carbon atoms, and l-dehydro derivatives thereof.

3. A -3,20-dioxc-9a-fiuoro 11o,l4e,17a.,21 tetrahydroxy-pregnadiene.

claim 3, said acyloxy group having from 1 to 10 carbon atoms 6. A-3,11,20-trioxo-9a-fiuoro-14a-hydroxy-pregnene.

References Cited in the file of this patent UNITED STATES PATENTS MurrayJuly 8, 1952 1 6 Haines Aug. 18, 1953 McNiven Aug. 31, 1954 ReichsteinDec. 4, 1956 Gould et a1. Feb. 26, 1957 Reichstein July 23, 1957 NobileJune 3, 1958 Hirschmann et a1 June 3, 1958 Herzog Sept. 30, 1958 Gouldet a1. Dec. 16, 1958 Bloom et a1. June 16, 1959

1. A COMPOUND OF THE FORMULA